Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 7 de 7
Filtrar
1.
Am J Nephrol ; 51(1): 43-53, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31822006

RESUMO

BACKGROUND: Renal biopsy is the mainstay of renal pathological diagnosis. Despite sophisticated diagnostic techniques, it is not always possible to make a precise pathological diagnosis. Our aim was to identify a genetic cause of disease in patients who had undergone renal biopsy and determine if genetic testing altered diagnosis or treatment. METHODS: Patients with suspected familial kidney disease underwent a variety of next-generation sequencing (NGS) strategies. The subset of these patients who had also undergone native kidney biopsy was identified. Histological specimens were reviewed by a consultant pathologist, and genetic and pathological diagnoses were compared. RESULTS: Seventy-five patients in 47 families underwent genetic sequencing and renal biopsy. Patients were grouped into 5 diagnostic categories based on pathological diagnosis: tubulointerstitial kidney disease (TIKD; n = 18); glomerulonephritis (GN; n = 15); focal segmental glomerulosclerosis and Alport Syndrome (n = 11); thrombotic microangiopathy (TMA; n = 17); and nonspecific pathological changes (n = 14). Thirty-nine patients (52%) in 21 families (45%) received a genetic diagnosis; 13 cases (72%) with TIKD, 4 (27%) with GN, 6 (55%) with focal segmental glomerulosclerosis/Alport syndrome, and 10 (59%) with TMA and 6 cases (43%) with nonspecific features. Genetic testing resulted in changes in understanding of disease mechanism in 21 individuals (54%) in 12 families (57%). Treatment would have been altered in at least 26% of cases (10/39). CONCLUSIONS: An accurate genetic diagnosis can result in changes in clinical diagnosis, understanding of pathological mechanism, and treatment. NGS should be considered as a complementary diagnostic technique to kidney biopsy in the evaluation of patients with kidney disease.


Assuntos
Testes Genéticos , Nefropatias/genética , Nefropatias/patologia , Rim/patologia , Adolescente , Adulto , Idoso , Biópsia , Criança , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Adulto Jovem
2.
Parasitol Res ; 107(5): 1257-64, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20676683

RESUMO

Albendazole is a benzimidazole drug which can be used to treat liver fluke (Fasciola hepatica) infections. Its mode of action is believed to be the inhibition of microtubule formation through binding to ß-tubulin. However, F. hepatica expresses at least six different isotypes of ß-tubulin, and this has confused, rather than clarified, understanding of the molecular mechanisms of benzimidazole drugs in this organism. Recombinant F. hepatica ß-tubulin proteins were expressed in, and purified from, Escherichia coli. These proteins were then used in pull-down assays in which albendazole was covalently linked to Sepharose. ß-Tubulin isotype 2 was pulled down in this assay, and this interaction could be reduced by adding competing albendazole. Molecular modelling of ß-tubulin isotypes suggests that changes in the side change conformations of residue 200 in the putative albendazole binding site may be important in determining whether, or not, a particular isotype will bind to the drug. These results, together with previous work demonstrating that albendazole causes disruption of microtubules in the liver fluke, strongly suggest that ß-tubulin isotype 2 is one of the targets of this drug.


Assuntos
Albendazol/metabolismo , Anti-Helmínticos/metabolismo , Fasciola hepatica/efeitos dos fármacos , Proteínas de Helminto/metabolismo , Tubulina (Proteína)/metabolismo , Animais , Sítios de Ligação , Escherichia coli/genética , Proteínas de Helminto/genética , Proteínas de Helminto/isolamento & purificação , Modelos Moleculares , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/isolamento & purificação , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Tubulina (Proteína)/genética , Tubulina (Proteína)/isolamento & purificação
3.
Biochem J ; 414(2): 177-87, 2008 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-18687061

RESUMO

The comparatively simple eukaryote Saccharomyces cerevisiae is composed of some 6000 individual genes. Specific sets of these genes can be transcribed co-ordinately in response to particular metabolic signals. The resultant integrated response to nutrient challenge allows the organism to survive and flourish in a variety of environmental conditions while minimal energy is expended upon the production of unnecessary proteins. The Zn(II)2Cys6 family of transcriptional regulators is composed of some 46 members in S. cerevisiae and many of these have been implicated in mediating transcriptional responses to specific nutrients. Gal4p, the archetypical member of this family, is responsible for the expression of the GAL genes when galactose is utilized as a carbon source. The regulation of Gal4p activity has been studied for many years, but we are still uncovering both nuances and fundamental control mechanisms that impinge on its function. In the present review, we describe the latest developments in the regulation of GAL gene expression and compare the mechanisms employed here with the molecular control of other Zn(II)2Cys6 transcriptional regulators. This reveals a wide array of protein-protein, protein-DNA and protein-nutrient interactions that are employed by this family of regulators.


Assuntos
Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transcrição Gênica , Galactose/metabolismo , Regulação Fúngica da Expressão Gênica , Modelos Biológicos , Estrutura Secundária de Proteína , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
4.
J Enzyme Inhib Med Chem ; 24(6): 1207-10, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19912053

RESUMO

The Escherichia coli protease inhibitor ecotin is believed to be implicated in the evasion of host defenses during infection. The protein has also attracted attention as a scaffold for the design of novel, specific protease inhibitors. Ecotin interacts with its targets through two sites. Key hydrophobic residues in both sites (Leu-64, Trp-67, Tyr-69, Met-84, and Met-85) were mutated to alanine and the effects on the inhibition of trypsin, chymotrypsin, and elastase were assessed. Each of these mutant ecotin proteins tested in kinetic assays with these enzymes exerted less inhibitory potency compared to wild-type ecotin. However, these effects were relatively small and not additive.


Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Proteínas Periplásmicas/química , Proteínas Periplásmicas/metabolismo , Inibidores de Serina Proteinase/metabolismo , Alanina/química , Alanina/genética , Alanina/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Substituição de Aminoácidos/fisiologia , Sítios de Ligação , Quimotripsina/antagonistas & inibidores , Quimotripsina/genética , Quimotripsina/metabolismo , Estabilidade Enzimática , Proteínas de Escherichia coli/genética , Cinética , Dados de Sequência Molecular , Mutação , Elastase Pancreática/antagonistas & inibidores , Elastase Pancreática/genética , Elastase Pancreática/metabolismo , Proteínas Periplásmicas/genética , Inibidores de Serina Proteinase/genética , Inibidores da Tripsina/genética , Inibidores da Tripsina/metabolismo
5.
Mol Biochem Parasitol ; 159(1): 73-8, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18372053

RESUMO

We have identified five alpha-tubulin and six beta-tubulin isotypes that are expressed in adult Fasciola hepatica. Amino acid sequence identities ranged between 72 and 95% for fluke alpha-tubulin and between 65 and 97% for beta-tubulin isotypes. Nucleotide sequence identity ranged between 68-77% and 62-80%, respectively, for their coding sequences. Phylogenetic analysis indicated that two of the alpha-tubulins and two of the beta-tubulins were distinctly divergent from the other trematode and nematode tubulin sequences described in this study, whereas the other isotypes segregated within the trematode clades. With regard to the proposed benzimidazole binding site on beta-tubulin, three of the fluke isotypes had tyrosine at position 200 of beta-tubulin, two had phenylalanine and one had leucine. All had phenylalanine at position 167 and glutamic acid at position 198. When isotype RT-PCR fragment sequences were compared between six individual flukes from the susceptible Cullompton isolate and from seven individual flukes from the two resistant isolates, Sligo and Oberon, these residues were conserved.


Assuntos
Fasciola hepatica/metabolismo , Tubulina (Proteína)/metabolismo , Sequência de Aminoácidos , Animais , Anti-Helmínticos/farmacologia , Benzimidazóis/farmacologia , Resistência a Medicamentos , Fasciola hepatica/efeitos dos fármacos , Fasciola hepatica/genética , Fasciola hepatica/crescimento & desenvolvimento , Dados de Sequência Molecular , Filogenia , Análise de Sequência de DNA , Tubulina (Proteína)/química , Tubulina (Proteína)/genética
6.
Int J Parasitol ; 43(14): 1133-9, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24162076

RESUMO

We have shown that Fasciola hepatica expresses at least six ß-tubulins in the adult stage of its life cycle, designated F.hep-ß-tub1-6 (Ryan et al., 2008). Here we show that different complements of tubulin isotypes are expressed in different tissues and at different life cycle stages; this information may inform the search for novel anthelmintics. The predominant (as judged by quantitative PCR) isotype transcribed at the adult stage was F.hep-ß-tub1 and immunolocalisation studies revealed that this isotype occurred mainly in mature spermatozoa and vitelline follicles. Quantitative PCR indicated that changes occurred in the transcription levels of ß-tubulin isotypes at certain life cycle stages and may be of importance in the efficacy of benzimidazole-based anthelmintic drugs, but there were no significant differences between the triclabendazole-susceptible Leon isolate and the triclabendazole-resistant Oberon isolate in the transcription levels of each of the isotypes. When three well-characterised isolates with differing susceptibilities to triclabendazole were compared, only one amino acid change resulting from a homozygous coding sequence difference (Gly269Ser) in isotype 4 was observed. However, this change was not predicted to alter the overall structure of the protein. In conclusion, these findings indicate that there is tissue-specific expression of tubulin isotypes in the liver fluke but the development of resistance to triclabendazole is not associated with changes in its presumed target molecule.


Assuntos
Fasciola hepatica/crescimento & desenvolvimento , Fasciola hepatica/genética , Regulação da Expressão Gênica no Desenvolvimento , Estágios do Ciclo de Vida , Tubulina (Proteína)/biossíntese , Tubulina (Proteína)/genética , Animais , Anti-Helmínticos/farmacologia , Benzimidazóis/farmacologia , Resistência a Medicamentos , Fasciola hepatica/efeitos dos fármacos , Perfilação da Expressão Gênica , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Reação em Cadeia da Polimerase em Tempo Real , Transcrição Gênica , Triclabendazol
7.
J Biol Chem ; 283(44): 30266-72, 2008 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-18701455

RESUMO

The GAL genes, which encode the enzymes required for normal galactose metabolism in yeast, are transcriptionally regulated by three proteins: Gal4p, an activator; Gal80p, an inhibitor; and Gal3p, a galactose sensor. These proteins control the switch between inert and active gene expression. The transcriptional activation function of Gal4p is rendered inactive in the presence of Gal80p. Here we present the three-dimensional structure of a complex between the acidic activation domain of Gal4p and Gal80p. The transactivation domain initiates with an extended region of polypeptide chain followed by two turns of an amphipathic alpha-helix. It fits into and across a deep cleft within the Gal80p dimer with the protein-protein interface defined primarily by hydrophobic interactions. A disordered loop in the apo-Gal80p structure (Asp-309 to Ser-316) becomes well-defined upon binding of the transactivation domain. This investigation provides a new molecular scaffold for understanding previous biochemical and genetic studies.


Assuntos
Kluyveromyces/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional , Sequência de Aminoácidos , Sítios de Ligação , Proteínas de Ligação a DNA , Espectroscopia de Ressonância Magnética , Modelos Biológicos , Conformação Molecular , Dados de Sequência Molecular , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Repressoras/química , Proteínas de Saccharomyces cerevisiae/química , Homologia de Sequência de Aminoácidos , Proteína Supressora de Tumor p53/química
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA